A total of 56 anterior blepharitis cases including 22 cases of ulcerative blepharitis and 34 cases of seborrheic blepharitis were studied. The predominant age group of anterior blepharitis cases was above 40 years. With males affected more than females. In the order of decreasing frequency, the isolated bacteria from anterior blepharitis in order of decreasing frequency were Staphylococcus aureus 14 (25%), Staphylococcus epidermidis 14 (25%), similar Klebsiella species 10 (18%), viridans Streptococci five (9%), Pseudomonas aeruginosa five (9%), Proteus species four (7%), Enterobacter aerugenes three (5%), and Escherichia coli one (2%). The common isolates observed in both samples were S. aureus, S. epidermidis, and Proteus species. The Gram-positive cocci S. aureus were resistant to four antibiotics and viridans Streptococci were resistant to three antibiotics, whereas the Gram-negative bacteria were resistant to two antibiotics. Improper selection of antibiotics, inadequate dosing, and poor compliance to therapy may play an important role in increasing resistance. Identification of anterior blepharitis pathogens and performing antibiotic susceptibility test are important factors in reducing the resistance to therapy.

Blepharitis is a common condition that causes inflammation of the eyelids. Sometimes referred to as granulated eyelids, blepharitis often produces flaky debris and particles at the base of the eyelashes. It can be acute or chronic, posing difficulties for both the patient and physician. There are three forms of blepharitis: Bacterial blepharitis, mainly staphylococcal, seborrheic blepharitis, and Meibomian gland More Details dysfunctional blepharitis. All the above forms are chronic in nature. [1]

Anatomically, the blepharitis can be divided into anterior and posterior blepharitis. The anterior blepharitis is broadly divided into bacterial blepharitis and seborrheic blepharitis, which reflects the underlying pathophysiology to a certain degree. Because there is often an overlap in a given individual, it is not unusual for the different entities to be difficult to distinguish clinically in primary care setting. [2],[3]

The pathophysiology of blepharitis is complex. It represents the interaction of various factors including abnormal lid margin secretions, lid margin bacteria, and dysfunctional precorneal tear film. [4] Staphylococcal blepharitis is characterized by scaling, crusting, and erythema of the eyelid margin with collaret formation at the base of the cilia. Chronic inflammation may be interrupted by acute exacerbations that lead to the development of ulcerative blepharitis, which is characterized by loss of eyelashes, corneal punctuates epithelial erosions, neovascularisation, and marginal infiltrates. [5] The bacterial exoenzymes destroy lipid molecules and release highly irritating free fatty acids, which in turn, disrupt the tear film integrity. [6]

Pathogenic bacteria cause ocular infection due to virulence and host's reduced resistance as a result of the factors like personal hygiene, living condition, socioeconomic status, and so forth. [7] The most common bacteria isolated from patients with chronic blepharitis are Staphylococcus aureus, Staphylococcus epidermidis, Propioibacterium acnes, and Corynebacteria. [5],[8]S. aureus and S. epidermidis were isolated with higher frequency (in 89-100% of cases) from the blepharitis cases, and their toxins have been reported to correlate with the presence of blepharoconjunctivitis. [9] Hence, there is a need for an immediate treatment for the serious infection that threatens even the cornea of eye. [10]

For specific antibacterial treatment, isolation and identification of bacterial pathogens along with antibiotics susceptibility pattern is essential. [11] There is a worldwide problem regarding the emergence of resistant strains toward antibiotics that have been routinely used in the hospitals. Bacterial pathogens, their antibiotic susceptibility, and resistance patterns may vary according to geographical and regional location. [12],[13] The bacterial sensitivity to various antibiotics varies from place to place and in the same place from time to time. Therefore, the changing spectrum of bacteria involved in ocular infections and the emergence of acquired microbial resistance dictate the need for continuous surveillance to guide empirical therapy. [2],[14]

The present study is to identify the spectrum of bacterial etiology of external ocular infections as well as to assess the sensitivity of the bacterial isolates to commonly prescribed antibiotics among patients in and around Misurata, Libya.

Materials and Methods

Sampling

This cross-sectional study included 56 anterior blepharitis samples such as ulcerative and seborrheic blepharitis for bacteriological evaluation from patients who were clinically diagnosed as anterior blepharitis at Al-Rahma ophthalmology clinic, Misurata, Libya, during the months of May 2012 to July 2012. Patients clinically diagnosed with anterior blepharitis and those who were willing to give information consent were included in the study. All patients were examined on the slit-lamp biomicroscope and diagnosed by a senior ophthalmologist. After detailed examination using standard techniques, [15] specimens were collected by senior ophthalmologist using sterilized moistened cotton swabs. In cases of ulcerative blepharitis, lashes deposit, tear film foaming content, and corneal punctuate erosions were swabbed, whereas in the seborrheic cases, only lashes deposit and acute hordeolum cyst were swabbed. All the swabbed cottons were immediately inoculated into a 2 ml of brain heart infusion broth (Scharlau, S.L. Barcelona) tubes and transferred to Misurata Medical college microbiology laboratory for further studies.

All the sample-inoculated brain heart infusion broth tubes were incubated at 37°C for 24-48 h. After incubation, the isolated bacterial cultures were further subcultured in blood agar, chocolate agar, mannitol salt agar, thioglycolate agar, and MacConkey's agar (Oxoid Ltd, England) plates. The inoculated media plates were incubated at their respective optimal temperature such as 37°C with 5-10% CO 2 for blood and chocolate agar plates, 37°C in aerobic conditions for mannitol salt agar and MacConkey's agar, and anaerobic condition for thioglycolate agar. [16] The plates were examined after 24 and 48 h and the bacterial isolates were identified up to species level based on their cultural characteristics and various biochemical-utilization patterns. [17] Bacterial colony appearance such as color, pigment production, and hemolysis on blood agar was observed and also identified by their Gram staining reactions. Further biochemical characterization of the isolates were performed by using oxidase, catalase, indole, methyl red, Voges-Proskauer, urease, citrate, and lactose fermentation for Gram-negative isolates, whereas catalase, coagulase, and hemolysin test were used for the identification of Gram-positive bacteria. [18]

A total of 56 patients were clinically diagnosed as anterior blepharitis, among that 22 cases are ulcerative blepharitis and 34 cases are seborrheic blepharitis. The predominant age group of anterior blepharitis cases was above 40 years. In this study, males are affected more than females. The demographic characteristics of the patients are summarized in [Table 1].

Table 1: Anterior blepharitis in relation to age and sex of sampled patients

In ulcerative blepharitis cases, S. aureus, viridians Streptococcus, and Pseudomonas aeruginosa were commonly isolated and S. epidermidis,Proteus species and Escherichia More Details coli were isolated in few cases. Klebsiella species and S. epidermidis were predominantly isolated in seborrheic blepharitis cases and also S. aureus, Enterobacter aerugenes, and Proteus species were isolated in few cases of seborrheic blepharitis. In both cases, S. epidermidis, S. aureus, and Proteus species were isolated. The isolated bacteria are shown in [Table 2].

Antibacterial susceptibility pattern of isolated bacteria was done on 12 commercially available antibiotic agents. [Table 3] and [Table 4] illustrate the susceptibility pattern of bacterial isolates from anterior blepharitis, including ulcerative and seborrheic blepharitis. Of the total number of bacterial isolates, 14 were S. aureus, 14 were S. epidermidis, 10 were Klebsiella species, five were viridans Streptococci, five were P. aeruginosa, four were Proteus species, three were E. aerugenes, and one was E. coli.

S. aureus showed high susceptibility to vancomycin, gentamycin, ciprofloxacin, and amikacin. On the other hand, they were highly resistant to ampicillin and moderately resistant to chloramphenicol, erythromycin, and cephalexin. Viridans Streptococci showed high susceptibility to vancomycin, chloramphenicol, ciprofloxacin, gentamycin, and doxycycline, whereas they were resistant to ampicillin and cephalexin. The S. epidermidis was highly susceptible to vancomycin, ciprofloxacin, gentamycin, and doxycycline.

The antimicrobial susceptibility pattern of Gram-negative bacteria is given in the [Table 4]. Klebsiella species showed high susceptibility to amikacin, ciprofloxacin, doxycycline, and streptomycin, and 70% strains were susceptible to ampicillin. The bacteria were moderately sensitive to chloramphenicol, clindamycin, and gentamycin. In all, 30% strains of Klebsiella species were resistant to cefuroxime, 20% strains were resistant to ampicillin, clindamycin, and gentamycin. P. aeruginosa were highly sensitive toward gentamycin, amikacin, and ciprofloxacin, erythromycin, doxycycline, and cefuroxime. A total of 60% strains of the P. aeruginosa were sensitive to chloramphenicol, whereas 40% strains were sensitive to cephalexin, clindamycin, streptomycin, and ampicillin. A total of 60% strains of the same bacteria were resistant to cephalexin and 40% strains were resistant to clindamycin and ampicillin. Proteus species also were highly susceptible to amikacin, gentamycin, ciprofloxacin, doxycycline, erythromycin, cefuroxime, and cephalexin. A total of 75% of the Proteus species were susceptibility to clindamycin and resistant to ampicillin.

E. aerugenes were completely susceptible to amikacin, gentamycin, clindamycin, chloramphenicol, doxycycline, streptomycin, and ciprofloxacin. A total of 67% strains of E. aerugenes were susceptible to ampicillin, erythromycin, cefuroxime and 33% resistant to cephalexin. E. coli were highly sensitive to ciprofloxacin, amikacin, erythromycin, clindamycin, and doxycycline; the bacteria showed intermediate activity to chloramphenicol, gentamycin, and streptomycin. The E. coli showed complete resistance to cefuroxime, cephalexin, and ampicillin.

Discussion

A total of 56 anterior blepharitis samples including 22 swabs from ulcerative blepharitis and 34 samples from seborrheic blepharitis. The predominant age group of anterior blepharitis cases was above 40 years, males were more than females and this may be related to occupational exposure.

The common isolates observed in both samples were S. aureus, S. epidermidis, and Proteus species. Similar studies conducted by Modarres et al., [20] Ramesh et al., [7] and Raju K. V [21] reported that S. aureus and S. epidermidis were predominant isolates from blepharitis patients. Staphylococcus species were isolated from maximum number of blepharitis samples by Tewelde et al., [21]Bharathi et al., [22] and Ubani et al.[10] High pathogenicity of S. aureus is attributed to their ability to multiply and spread widely in tissues through their production of many extracellular substances like coagulase, which deposits fibrin on the surface of the microorganism altering their phagocytosis, and alpha toxin (hemolysin), which lyses erythrocyte and damages platelets.

Viridans Streptococcus and P. aeruginosa were second frequently isolated bacteria from the ulcerative blepharitis; the P. aeruginosa may transfer mechanically and produce exotoxin-A, which causes tissue necrosis and ulceration. [23]Proteus species and E. coli were isolated in very few cases. In contrast, Pseudomonas strains were more often isolated in ulcerative blepharitis by Cruz CS et al., [24] in Ghana [25] and in Hong Kong. [26] Like this viridans Streptococci were often isolated in blepharitis reported in many studies. [27],[28],[29]

Klebsiella species were second frequently isolated bacteria from seborrheic blepharitis. Proteus species were isolated in few cases of both blepharitis, E. coli and Enterobacter aerogenes were isolated from only one case of ulcerative and seborrheic blepharitis. Destan N K et al., [30] also isolated E. coli, Proteus mirabilis, and Enterobacter aerogenes from few cases of bacterial blepharitis infections and also the similar results were observed in Nigeria [6] and Israel. [31]

In our study S. aureus, S. epidermidis, and Klebsiella species were predominant bacteria. There are few differences like the prevalence and degree of occurrence in our study in comparison with reports.

Based on antibiotic susceptibility pattern, the Gram-positive cocci S. aureus,S. epidermidis, and viridans Streptococci were highly susceptible to vancomycin, ciprofloxacin, gentamycin, and doxycycline. S. aureus and S. epidermidis were highly susceptible to amikacin, and the viridans Streptococci were also susceptible to chloramphenicol. S. aureus were moderately resistant to chloramphenicol, erythromycin, and cephalexin, whereas in the study done by Ahmed M O et al., [32] isolated S. aureus in Libya were 74% resistant to erythromycin. Khosravi A D et al., [14] also isolated S. aureus that were resistant to vancomycin. Viridans Streptococci were resistant to ampicillin and cephalexin. This finding is in agreement with studies conducted in Ethiopia, [33] India, [21],[34] Iron, [14] and Nigeria. [10]

Gram-negative bacteria were highly susceptible to amikacin, gentamycin, ciprofloxacin, erythromycin, and doxycycline. Cefuroxime had shown moderate activity against Klebsiellaspecies and E.aerugenes. Streptomycin had shown good efficacy against Klebsiella and E. aerugenes. Clindamycin showed moderate activity against P. aeruginosa, Klebsiella species, and Proteus species. More than 50% strains of Proteus species and P. aeruginosa were resistant to ampicillin. Khalifa S G et al., [35] isolated E. coli, Klebsiella species, Enterobacter species, and Proteus species in Libya, the above isolates were 57.7% resistant to ampicillin, 25.6% to chloramphenicol, and 6% to gentamycin. The E. coli species were also completely resistant to ampicillin, cefuroxime, and cephalexin. In all, 20% strains of Klebsiella species were resistant to ampicillin, cefuroxime, chloramphenicol, clindamycin, and ciprofloxacin. Khalifa G S et al., [35] also found out cefuroxime-resistant E. coli and Klebsiella species.

Pattern of antibiotic susceptibility may vary in different geographical areas. [21] The problem of antibiotic resistance is very serious in Libya and appears to be on the rise. High resistance rates were observed among Gram-negative bacteria against commonly used drugs (i.e., ampicillin, trimethoprim-sulfamethoxazole, and cephalosporins). [35] An attempt should be made for identification of anterior blepharitis pathogens and perform antibiotic susceptibility tests. The susceptibility tests are in vitro results and do not always mirror the clinical response to antibiotics due to a variety of reasons including direct topical delivery, corneal penetration of an antibiotic, and host factors. [36]

Conclusion

Improper selection of antibiotics, inadequate dosing, and poor compliance to therapy may play an important role in increasing resistance. However, the information provided in this study will aid the clinician to make a decision about the empirical antibiotic treatment of bacterial ocular infections that cause major public health problems.